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Linking microbial community structure and microbial processes: An empirical and conceptual overview

A major goal of microbial ecology is to identify links between microbial community structure and microbial processes. Although this objective seems straightforward, there are conceptual and methodological challenges to designing studies that explicitly evaluate this link. Here, we analyzed literature documenting structure and process responses to manipulations to determine the frequency of structure-process links and whether experimental approaches and techniques influence link detection. We examined nine journals (published 2009–13) and retained 148 experimental studies measuring microbial community structure and processes. Many qualifying papers (112 of 148) documented structure and process responses, but few (38 of 112 papers) reported statistically testing for a link. Of these tested links, 75% were significant and typically used Spearman or Pearson's correlation analysis (68%). No particular approach for characterizing structure or processes was more likely to produce significant links. Process responses were detected earlier on average than responses in structure or both structure and process. Together, our findings suggest that few publications report statistically testing structure-process links. However, when links are tested for they often occur but share few commonalities in the processes or structures that were linked and the techniques used for measuring them.

FEMS Microbiology Ecology

Mineral stimulation of subsurface microorganisms: release of limiting nutrients from silicates

Microorganisms play an important role in the weathering of silicate minerals in many subsurface environments, but an unanswered question is whether the mineral plays an important role in the microbial ecology. Silicate minerals often contain nutrients necessary for microbial growth, but whether the microbial community benefits from their release during weathering is unclear. In this study, we used field and laboratory approaches to investigate microbial interactions with minerals and glasses containing beneficial nutrients and metals. Field experiments from a petroleum-contaminated aquifer, where silicate weathering is substantially accelerated in the contaminated zone, revealed that phosphorus (P) and iron (Fe)-bearing silicate glasses were preferentially colonized and weathered, while glasses without these elements were typically barren of colonizing microorganisms, corroborating previous studies using feldspars. In laboratory studies, we investigated microbial weathering of silicates and the release of nutrients using a model ligand-promoted pathway. A metal-chelating organic ligand 3,4 dihydroxybenzoic acid (3,4 DHBA) was used as a source of chelated ferric iron, and a carbon source, to investigate mineral weathering rate and microbial metabolism. In the investigated aquifer, we hypothesize that microbes produce organic ligands to chelate metals, particularly Fe, for metabolic processes and also form stable complexes with Al and occasionally with Si. Further, the concentration of these ligands is apparently sufficient near an attached microorganism to destroy the silicate framework while releasing the nutrient of interest. In microcosms containing silicates and glasses with trace phosphate mineral inclusions, microbial biomass increased, indicating that the microbial community can use silicate-bound phosphate inclusions. The addition of a native microbial consortium to microcosms containing silicates or glasses with iron oxide inclusions correlated to accelerated weathering and release of Si into solution as well as the accelerated degradation of the model substrate 3,4 DHBA. We propose that silicate-bound P and Fe inclusions are bioavailable, and microorganisms may use organic ligands to dissolve the silicate matrix and access these otherwise limiting nutrients.

Chemical Geology

Linking microbial and ecosystem ecology using ecological stoichiometry: a synthesis of conceptual and empirical approaches

Currently, one of the biggest challenges in microbial and ecosystem ecology is to develop conceptual models that organize the growing body of information on environmental microbiology into a clear mechanistic framework with a direct link to ecosystem processes. Doing so will enable development of testable hypotheses to better direct future research and increase understanding of key constraints on biogeochemical networks. Although the understanding of phenotypic and genotypic diversity of microorganisms in the environment is rapidly accumulating, how controls on microbial physiology ultimately affect biogeochemical fluxes remains poorly understood. We propose that insight into constraints on biogeochemical cycles can be achieved by a more rigorous evaluation of microbial community biomass composition within the context of ecological stoichiometry. Multiple recent studies have pointed to microbial biomass stoichiometry as an important determinant of when microorganisms retain or recycle mineral nutrients. We identify the relevant cellular components that most likely drive changes in microbial biomass stoichiometry by defining a conceptual model rooted in ecological stoichiometry. More importantly, we show how X-ray microanalysis (XRMA), nanoscale secondary ion mass spectroscopy (NanoSIMS), Raman microspectroscopy, and in situ hybridization techniques (for example, FISH) can be applied in concert to allow for direct empirical evaluation of the proposed conceptual framework. This approach links an important piece of the ecological literature, ecological stoichiometry, with the molecular front of the microbial revolution, in an attempt to provide new insight into how microbial physiology could constrain ecosystem processes.

Ecosystems

Microbial responses and nitrous oxide emissions during wetting and drying of organically and conventionally managed soil under tomatoes

The types and amounts of carbon (C) and nitrogen (N) inputs, as well as irrigation management are likely to influence gaseous emissions and microbial ecology of agricultural soil. Carbon dioxide (CO2) and nitrous oxide (N2O) efflux, with and without acetylene inhibition, inorganic N, and microbial biomass C were measured after irrigation or simulated rainfall in two agricultural fields under tomatoes (Lycopersicon esculentum). The two fields, located in the California Central Valley, had either a history of high organic matter (OM) inputs ("organic" management) or one of low OM and inorganic fertilizer inputs ("conventional" management). In microcosms, where short-term microbial responses to wetting and drying were studied, the highest CO2 efflux took place at about 60% water-filled pore space (WFPS). At this moisture level, phospholipid fatty acids (PLFA) indicative of microbial nutrient availability were elevated and a PLFA stress indicator was depressed, suggesting peak microbial activity. The highest N 2O efflux in the organically managed soil (0.94 mg N2O-N m-2 h-1) occurred after manure and legume cover crop incorporation, and in the conventionally managed soil (2.12 mg N2O-N m-2 h-1) after inorganic N fertilizer inputs. Elevated N2O emissions occurred at a WFPS >60% and lasted <2 days after wetting, probably because the top layer (0-150 mm) of this silt loam soil dried quickly. Therefore, in these cropping systems, irrigation management might control the duration of elevated N2O efflux, even when C and inorganic N availability are high, whereas inorganic N concentrations should be kept low during times when soil moisture cannot be controlled.

Biology and Fertility of Soils

Understanding how microbiomes influence the systems they inhabit

Translating the ever-increasing wealth of information on microbiomes (environment, host, or built environment) to advance the understanding of system-level processes is proving to be an exceptional research challenge. One reason for this challenge is that relationships between characteristics of microbiomes and the system-level processes they influence are often evaluated in the absence of a robust conceptual framework and reported without elucidating the underlying causal mechanisms. The reliance on correlative approaches limits the potential to expand the inference of a single relationship to additional systems and advance the field. We propose that research focused on how microbiomes influence the systems they inhabit should work within a common framework and target known microbial processes that contribute to the system-level processes of interest. Here we identify three distinct categories of microbiome characteristics (microbial processes, microbial community properties, and microbial membership) and propose a framework to empirically link each of these categories to each other and the broader system level processes they affect. We posit that it is particularly important to distinguish microbial community properties that can be predicted from constituent taxa (community aggregated traits) from and those properties that are currently unable to be predicted from constituent taxa (emergent properties). Existing methods in microbial ecology can be applied to more explicitly elucidate properties within each of these categories and connect these three categories of microbial characteristics with each other. We view this proposed framework, gleaned from a breadth of research on environmental microbiomes and ecosystem processes, as a promising pathway with the potential to advance discovery and understanding across a broad range of microbiome science.

Nature Microbiology

Compositional analysis of formation water geochemistry and microbiology of commercial and carbon dioxide-rich wells in the southwestern United States

Studies of naturally occurring subsurface carbon dioxide (CO 2 ) accumulations can provide useful information for potential CO 2 injection projects; however, the microbial communities and formation water geochemistry of most reservoirs are understudied. Formation water and microbial biomass were sampled at four CO 2 -rich reservoir sites: two within Bravo Dome, a commercial CO 2 field in New Mexico; one northwest of Bravo Dome in Colorado (Oakdale Field); and one southwest of Bravo Dome in New Mexico (Rafter “K” Ranch). Aside from the Rafter “K” Ranch site, minor differences were observed in the geochemistry of formation water collected from these sites compared to historical data. No organisms were significantly associated with Oakdale Field compared to the other three sites, nor were any hydrogeochemical or gas geochemical parameters (for example, CO 2 concentration) found to have significant associations with the microbial ecology of these four sites. Microorganisms from these sites were metabolically diverse and had the potential to (1) generate methane, (2) produce corrosive hydrogen sulfide (H 2 S), and (3) rapidly biofoul and (or) clog pore spaces by shifting microbial communities with changes in salinity or nutrient supply. This study demonstrates that high concentrations of CO 2 in subsurface reservoirs apparently have not imparted a distinct geochemical or microbiological signature on the associated formation waters and that the microorganisms in these reservoirs are metabolically diverse and could adapt to geochemical changes in the subsurface.

Colorado, New Mexico, Texas, Oklahoma

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arx A was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arx A-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Environmental Microbiology

Hydraulic connectivity and hydrochemistry influence microbial community structure in agriculturally-affected alluvial aquifers in the Midwestern United States

Alluvial aquifers can provide ecosystem services and drinking water, but much remains unknown about human effects on aquifer microbiomes. Therefore, we used amplicon sequencing and hydrochemical characterization to pair microbial communities with environmental conditions across 37 alluvial aquifer wells. The study region spanned eastern Iowa and southern Minnesota (USA) and contained a combination of drinking water and monitoring wells. In terms of microbial ecology, dominant phyla across the wells included Proteobacteria, Bacteroidota, Patescibacteria, Planctomycetota, and Nitrospirota. Tritium, an indicator of infiltration and surface water influence, was the highest correlated variable with the Shannon index (α-diversity) by the Spearman rank sum (ρ = 0.60) and one of only four significant environmental variables in the constrained correspondence analysis. We built random forest regression models to predict tritium concentrations from microbial family relative abundance (held-out testing coefficient of determination ( R 2 ) = 0.77 and mean absolute percentage error = 7%) and interpreted the models with Shapley additive explanation values. The most important families for predicting tritium concentrations were Nitrosopumilaceae and Methylomirabilaceae . Upwelling methane could contribute to the unusual coupling of ammonia oxidation by Nitrosopumilaceae with simultaneous nitrite-dependent methane oxidation by Methylomirabilaceae . Taken together, we illuminate the relationship among hydrochemistry, hydraulic connectivity, and alluvial aquifer microbiomes.

Iowa, Minnesota

Microbial transformation of elements: The case of arsenic and selenium

Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table. These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles. Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized. Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations. Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e. the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g. FeNiSe hydrogenase). Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC). The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria. Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments. The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g. convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements.

International Microbiology

Biogeochemistry of aquifer systems

Many studies have examined the differences in bacterial numbers, composition, and activity between groundwater and sediment samples. The majority of the literature has suggested higher percentages of attached bacteria than of unattached bacteria in aquifer systems, including in pristine aquifers and in aquifers contaminated with petroleum, creosote, sewage, and landfill leachate. In studies of aquifer biogeochemistry, much useful information regarding the microbial ecology of the system can be obtained by looking at organic compound and electron acceptor concentrations. An overview of approaches for identifying the redox characteristics of sediment is given in Christensen et al., and methods specific for determining reactive iron species in aquifers are reviewed by Heron et al. and Tuccillo et al. Other solid-phase electron acceptors that are important in aquifer systems include Mn(IV) oxides and barite. Important biogeochemical reactions catalyzed by indigenous microorganisms also are studied using a variety of experimental approaches including laboratory batch and column experiments as well as field-based in situ microcosms, tracer tests, and push-pull tests. The advantage of using a radiolabeled tracer in a study was that the reaction rates could be determined for the different steps in the denitrification pathway. Historically, researchers trained in geochemistry and hydrology created and tested hypotheses about aquifer biogeochemistry through laboratory assays and field-based geo-chemical measurements and experiments. Jeon et al. extended this research by using push-pull tests combined with stable-isotope probing to identify the specific members of the microbial community actively degrading naphthalene and rates of naphthalene degradation.

Book chapter

Using transport model interpretations of tracer tests to study microbial processes in groundwater

It has long been known that microorganisms affect the geochemistry of groundwater. But despite this recognition, little detailed information is available regarding the rates and the factors controlling microbial processes in groundwater. Part of the reason stems from the relatively inaccessible nature of most groundwater and the difficulties encountered in obtaining representative samples of groundwater and subsurface sediments. At the same time, most groundwater systems are nutrient poor or oligotrophic environments in which the resident microorganisms are severely stressed and often nearly inactive. These populations are functioning so slowly that many types of activity measurements designed to assess microbial processes in more productive environments are ineffective for groundwater. However, because groundwater is by far the largest reservoir of freshwater in the world ( Freeze and Cherry 1979 ), our lack of knowledge about groundwater microorganisms and their processes represents a significant void in the study of microbial ecology and in our ability to predict the outcome when these reserves are compromised by contamination.

Book chapter

Stress tolerance in plants via habitat-adapted symbiosis

We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.The ISME Journal (2008) 2, 404-416; doi:10.1038/ismej.2007.106; published online 7 February 2008. ?? 2008 International Society for Microbial Ecology All rights reserved.

ISME Journal

Advances in the hydrogeochemistry and microbiology of acid mine waters

The last decade has witnessed a plethora of research related to the hydrogeochemistry and microbiology of acid mine waters and associated tailings and waste-rock waters. Numerous books, reviews, technical papers, and proceedings have been published that examine the complex bio-geochemical process of sulfide mineral oxidation, develop and apply geochemical models to site characterization, and characterize the microbial ecology of these environments. This review summarizes many of these recent works, and provides references for those investigating this field. Comparisons of measured versus calculated Eh and measured versus calculated pH for water samples from several field sites demonstrate the reliability of some current geochemical models for aqueous speciation and mass balances. Geochemical models are not, however, used to predict accurately time-dependent processes but to improve our understanding of these systems and to constrain possible processes that contribute to actual or potential water quality issues. Microbiological studies are demonstrating that there is much we have yet to learn about the types of different microorganisms and their function and ecology in mine-waste environments. A broad diversity of green algae, bacteria, archaea, yeasts, and fungi are encountered in acid mine waters, and a better understanding of their ecology and function may potentially enhance remediation possibilities as well as our understanding of the evolution of life.

International Geology Review

Microbial consortia of gorgonian corals from the Aleutian islands

Gorgonians make up the majority of corals in the Aleutian archipelago and provide critical fish habitat in areas of economically important fisheries. The microbial ecology of the deep-sea gorgonian corals Paragorgea arborea, Plumarella superba , and Cryogorgia koolsae was examined with culture-based and 16S rRNA gene-based techniques. Six coral colonies (two per species) were collected. Samples from all corals were cultured, and clone libraries were constructed from P. superba and C. koolsae . Cultured bacteria were dominated by the Gammaproteobacteria , especially Vibrionaceae , with other phyla comprising <6% of the isolates. The clone libraries showed dramatically different bacterial communities between corals of the same species collected at different sites, with no clear pattern of conserved bacterial consortia. Two of the clone libraries (one from each coral species) were dominated by Tenericutes , with Alphaproteobacteria dominating the remaining sequences. The other libraries were more diverse and had a more even distribution of bacterial phyla, showing more similarity between genera than within coral species. Here we report the first microbiological characterization of P. arborea, P. superba , and C. koolsae .

Alaska

The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures

The interdependence between geoelectrical signatures at underground petroleum plumes and the structures of subsurface microbial communities was investigated. For sediments contaminated with light non-aqueousphase liquids, anomalous high conductivity values have been observed. Vertical changes in the geoelectrical properties of the sediments were concomitant with significant changes in the microbial community structures as determined by the construction and evaluation of 16S rRNA gene libraries. DNA sequencing of clones from four 16S rRNA gene libraries from different depths of a contaminated field site and two libraries from an uncontaminated background site revealed spatial heterogeneity in the microbial community structures. Correspondence analysis showed that the presence of distinct microbial populations, including the various hydrocarbon-degrading, syntrophic, sulfate-reducing, and dissimilatory-iron-reducing populations, was a contributing factor to the elevated geoelectrical measurements. Thus, through their growth and metabolic activities, microbial populations that have adapted to the use of petroleum as a carbon source can strongly influence their geophysical surroundings. Since changes in the geophysical properties of contaminated sediments parallel changes in the microbial community compositions, it is suggested that geoelectrical measurements can be a cost-efficient tool to guide microbiological sampling for microbial ecology studies during the monitoring of natural or engineered bioremediation processes. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

Gulf of Mexico blue hole harbors high levels of novel microbial lineages

Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic and geochemical characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the hole’s rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40–75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80–110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

Interational Society of Microbial Ecology (ISME) J

Covariance of bacterioplankton composition and environmental variables in a temperate delta system

We examined seasonal and spatial variation in bacterioplankton composition in the Sacramento-San Joaquin River Delta (CA) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Cloned 16S rRNA genes from this system were used for putative identification of taxa dominating the T-RFLP profiles. Both cloning and T-RFLP analysis indicated that Actinobacteria , Verrucomicrobia , Cytophaga-Flavobacterium and Proteobacteria were the most abundant bacterioplankton groups in the Delta. Despite the broad variety of sampled habitats (deep water channels, lakes, marshes, agricultural drains, freshwater and brackish areas), and the spatial and temporal differences in hydrology, temperature and water chemistry among the sampling campaigns, T-RFLP electropherograms from all samples were similar, indicating that the same bacterioplankton phylotypes dominated in the various habitats of the Delta throughout the year. However, principal component analysis (PCA) and partial least-squares regression (PLS) of T-RFLP profiles revealed consistent grouping of samples on a seasonal, but not a spatial, basis. b- Proteobacteria related to Ralstonia , Actinobacteria related to Microthrix , and b- Proteobacteria identical to the environmental Clone LD12 had the highest relative abundance in summer/fall T-RFLP profiles and were associated with low river flow, high pH, and a number of optical and chemical characteristics of dissolved organic carbon (DOC) indicative of an increased proportion of phytoplankton-produced organic material as opposed to allochthonous, terrestrially derived organic material. On the other hand, Geobacter -related b- Proteobacteria showed a relative increase in abundance in T-RFLP analysis during winter/spring, and probably were washed out from watershed soils or sediment. Various phylotypes associated with the same phylogenetic division, based on tentative identification of T-RFLP fragments, exhibited diverse seasonal patterns, suggesting that ecological roles of Delta bacterioplankton were partitioned at the genus or species level.

Aquatic Microbial Ecology

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum , and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active “ Chlorobi PRB” MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. “ M. paradoxum PRB” encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

Montana, Wyoming